Illumina MiSeq paired end sequencing; RDA-seq (Next generation sequencing of Representational Difference Analysis products) of wheat lodicules
RDA-seq (Next generation sequencing of Representational Difference Analysis products) of wheat lodicules
The plant material consisted of two highly-chasmogamous (HCH) cultivars: Piko (DE, 2009, CWW-3319.5/3/Kraka//Maris Huntsman/Fruhgold) and Poezja (LT, 2017, STH43/STH248-93-14-326), and two low-chasmogamy (LCH) cultivars: Euforia (PL, 2018, Muszelka//N5113/STH3907) and KWS Dacanto (PL, 2011, Opus/Certo) cultivars of hexaploid winter wheat. In order to identify candidate genes controlling lodicules opening, lodicules of HCH and LCH genotypes were harvested from spikes at pre-flowering (GS95 - ear completely emerged above flag leaf ligule) corresponding to green anther stage (GAS) and early flowering (GS61 - start of flowering (first anthers visible) referred as yellow anther stage (YAS) (5, 32). The samples were collected in three biological replicates. Approximately 50-100 lodicules were samples from five to seven individual spikes for each of the three replicates. The materials were immediately submerged in RNAlater™ Stabilization Solution (Ambion, Thermo Fisher Scientific, Waltham, MA, USA) and kept at -80˚C until required.  After 6-week vernalization at 4˚C, plants were transferred to a growth chamber (ForClean, ZalMed Sp. z.o.o, Warsaw, Poland) under the following conditions: temperature - 18˚C/15˚C day/night, 14/10 h photoperiod and constant relative humidity of 50%. Plants were cultivated in pots filled with a mixture of peat and perlite (1:1, v/v), three plants per single pot (19 cm diameter). The plants were hand irrigated daily to maintain soil water content close to field capacity. GeneMATRIX Universal RNA Purification kit (EURx, Gdańsk, Poland) was used to isolate total RNA from the preserved lodicules. RNA concentration was measured by means of a Qubit® 2.0 Fluorometer (Invitrogen, Waltham, MA, USA) and its quality and integrity were verified on BioAnalyzer 2100 (Agilent Technologies, Santa Clara, CA, USA) using Agilent RNA 6000 Nano Kit.  The first and the second strand of cDNA smART were obtained using First Strand cDNA Synthesis Kit (EURx, Gdańsk, Poland) and Second Strand cDNA Synthesis Kit (abm, Richmond, BC, Canada), respectively. Representational difference analysis was performed on high quality cDNA according to Hubank and Schatz (1994) procedure with small modifications. Lodicules at the pre-flowering were used as testers while the lodicules early-flowering were selected as driver probes. The analysis was taken in three replicates for each variety. The tester:driver ratio in three subsequent rounds of subtractive hybridization increased from 1:50, and 1:400 to 1:200000. Differential products obtained in the third subtraction of the size range from 200 to 800 bp were sequenced. Nextera DNA Flex Library Prep Kit (Illumina, San Diego, CA, USA) was used for library construction according to the manufacturer instructions.